Fast-Track and Integration-Free Method of Genome Editing by CRISPR/Cas9 in Murine Pluripotent Stem Cells

The CRISPR/Cas9 system has unprecedentedly revolutionized genome-editing technology, which is being successfully applied virtually in all branches of biological sciences. Although much success has been attained in gene manipulation, still the majority of methods are laborious and non-integration-free, and require prolonged time for the expansion of mutant cell pools/clones, while fewer cells exhibit functional knockout efficiency. To overcome these obstacles, here, we describe an efficient, inexpensive, integration-free, and rapid one-step protocol for CRISPR/Cas9-assisted gene knockout in murine pluripotent stem cells (PSCs). Our protocol has streamlined both the liposome-based transfection system and screening strategy to work more efficiently with small numbers of PSCs (∼2.0 × 104 cells) and to minimize laborious steps of lentiviral packaging, transduction, and single-clone passaging. In our method, around 90% (CI = 95%, 79.5230%–100%) of PSC colonies harbored functional knockout in the context of protein expression. Therefore, the current protocol is technically feasible, time-saving, and highly efficient for genome editing in pluripotent stem cells.

i. Prepare LB buffer and autoclave at 121 0 C for 15 min ( Table 2). The LB buffer can be stored at 4 0 C.
Note: Add ampicillin to the LB buffer before use (100 μg/mL).
ii. To prepare agar plate, add 15g agar powder to 1LB and autoclave at 121 0 (Table 2). Cool adequately, add ampicillin (100 μg/mL) and keep it at room temperature to solidify. The LB buffer can be stored at 4 0 C for around 2 months.  ii. Check the specificity of a sgRNA using 20nt gRNA sequence plus PAM motif NGG CACC and AAAC (black) following BsmBI digestion of pLentiCRISPR V2 are added to the 5′ end of each sgRNA primer respectively. G (red) after the overhang CACC is added to the 5′ region of forward sgRNA, and its complementary C (red) is added to the 3′ end of the reverse sgRNA primer because U6 promoter requires G to express the sgRNA. Copy and paste the sgRNAs of the desired gene (Mdm2, Stub1 and Yy1) separately into the highlighted region (Table 4).  CCGG and AAAC (black) following Bsa I digestion of pGL3-U6-sgRNA-PGK-puromycin were added to the 5′ end of each sgRNA primer respectively. G (red) after the overhang CACC is added to the 5′ region of forward sgRNA, and its complementary C (red) is added to the 3′ end of the reverse sgRNA primer because U6 promoter requires G to express the sgRNA. Copy and paste the sgRNAs of the desired gene separately into the highlighted region (Table 5). Note. In fact, de-salted standard oligos are sufficient for efficient cloning.

Preparation of single and two-vector constructs
Timing: 2 days

Annealing oligo pair:
a. Forward and reverse oligonucleotides of sgRNA were dissolved in ddH2O and diluted to a final concentration of 10μM. The oligos were mixed as follow:
b. The oligo mix can be annealed by heating at 95 0 C for 5 minutes and cooling at room temperature (~25 0 C).
e. Cut the gel containing larger fragments for plentiCRISPRv2 or a single fragment of pGL3-U6-sgRNA-PGK-puromycin and purify the digested plasmid using GeneJET gel extraction kit.
Dissolve the extracted plasmid into 10μl ddH2O. c. Add the sgRNA-lentiCRISPRv2 ligation reaction mix/ pGL3-U6-sgRNA-PGK-puromycin ligation into an Eppendorf tube containing Trans5α chemically competent cells, incubate the mixture on ice for 30 min, followed by heat shock at 42 0 C water bath for the 90s, and immediate re-incubation on ice for 5 min.

Note:
To reduce the chances of potential homologous recombination, the transformation of lentiviral plasmids into recombination-deficient bacteria (e.g., Stbl3) is recommended.
d. Add 50-100μl LB to the ligation-bacteria mix from step 2.3.c and incubate the mixture at 37 0 C for 45 minutes in a shaker.
e. Spread the mixture from step 2.3.d onto an agar LB dish (ampicillin) and incubate at 37 0 C for 1 day.
g. Extract plasmid DNA from the bacteria using GeneJET plasmid miniprep kit as per manufacturer's instruction.

Note:
The 20bp gRNA sequence should be placed between the U6 promoter and the remainder of the gRNA scaffold in the plentiCRISPR v2 construct. Table 6. List of PCR primers.